In vitro cultured cells, including mammalian cells, are extensively used in cell biology studies. Until the middle 80's, cell culture techniques were labor-intensive and did not scale to high cell numbers. Currently, there is a wide acceptance and universal application of High Throughput Screening (HTS) using cell-based assays in the Pharmaceutical and Biotechnology industry as well as in basic research.
HTS screening is performed in microplates. The surface of these microplates may be modified for cell culturing applications, typically using a plasma discharge for easier cell attachment. These cell culture microplates have eased larger scale cultivation of cells impacting both drug discovery and manufacturing. With respect to drug discovery, cell-based assays are increasingly being used for drug target validation in efficacy studies using high content analyses, and also in vitro ADMET (drug absorption, distribution, metabolism, elimination and toxicity). These studies are performed on cells as they provide more representative responses to drugs than simple molecular assays, and are easier to use in a high-throughput format than animals.
Cryopreservation is the gold standard method for long term storage of cultured cells. Since cryopreservation can adversely affect the viability and function of cells, methodological freezing and thawing procedures have been developed in order to preserve the correct structure, function, behaviour and biology of cells in culture. In early years, the addition of cryoprotective agents including glycerol and dimethyl sulphoxide (DMSO) were already used both for the preservation of tumour cells and also for healthy hematopoietic primary cells. In general, current standards indicate that long term frozen cells are stored at −130° C. or below in liquid nitrogen vapour phase to ensure the highest level of viability. For shorter periods of time commonly used for transportation purposes (usually from 1 to 3 days), frozen cells can be stored between −70° C. and −80° C.
Frozen cells may be contained in straws or, more commonly, in ampoules of 1 ml to 5 ml also known as cryovials or cryotubes, with a 10% of cryopreservant.
Basic thawing conditions are already established in the art as beneficial for the recovery of the cells. These thawing conditions remain as current standard protocols and include thawing temperature optimization, dilution of cryopreserved solution by adding cell culture medium with serum or complete cryoprotective agent removal by centrifugation. These standard thawing protocols (e.g. ATCC protocols) indicate that the cryovial has to be thawed rapidly by placing it in a water bath at 37° C. and continuous agitation is applied until its contents have been thawed completely. Then, the vial is decontaminated and the content is immediately transferred to a culture vessel containing normally up to 10 volumes of appropriate culture medium. This dilution is performed drop-wise in order to minimize as possible the osmotic shock. It will lower the cryoprotective agent's concentration (commonly DMSO) to a level that does not need its immediate complete removal for most cells. The entire thawing operation must be done as fast as possible in order to minimize the toxic effects of the cryoprotective agent to the cells. Upon cell attachment, commonly within the next 24 hours of incubation at 37° C. in a 5% CO2 atmosphere, the medium is replaced with a fresh one and that expedites the removal of the cryoprotective agent. For cell types more sensitive to the cryoprotective agent, centrifugation must be performed before seeding in order to completely remove the toxic agent.
Cell cultures are typically expanded in vitro and harvested at a confluence of 80% or more. Several methods are used in the art for harvesting cell cultures, including enzymatic digestion using trypsin and EDTA, mechanical lifting using cell scrapers, or temperature responsive surfaces i.e. UpCell™ from Nunc. After cell harvesting, cells are commonly centrifuged, diluted, counted using a haemocytometer or an automatic cell counter and either seeded again or suspended at the right concentration of cryoprotective agent.
Cells are seeded in regular cell culture vessels also called flasks, grown in vitro for few days and then re-seeded into microplates when high-throughput use is required. This tissue culture process is still the current standard for seeding cells in a high throughput format, being a laborious and time consuming method that requires proper cell culture facilities and qualified technicians. The result is exposed to microbial contamination and cross-contamination of the cells due to the numerous handling procedures involved.
JR 2002253205 A has tried to address this issue by directly freezing down cells that are adhered to a plate. US 2002012901 A1 teaches about thawing methods also related to adherent frozen cells. However, it is well known that cells frozen in monolayers are more susceptible to freezing injury than those in suspension. It has been reported that it might be due to the presence of gap junctions that ease the spread of ice between neighboring cells (Armitage et al. Cryobiology 2003; Liu et al. Cryo letters 2003, Acker J P et al., 2001). JR 2069200 A and CA 2689946 A1 teach about adherent frozen cells on plates and just focused on diagnostic tests to reveal intracellular parasites and viruses. None of these methods teach about several-layer frozen vials as per in the present invention.
WO 2006072335 A1 and EP 1869976 are considered the closest prior art publications, teaching about suspended cells frozen in muitiwell plates at an oxygen partial pressure lower than atmospheric pressure at the time of cell freezing in order to extend their shelf life. None of these two publications teach about higher-efficiency several-layer frozen vials, and these methods need indeed to dilute by the addition of culture medium to the thawed cells before performing any cell-based assay, in order to avoid the toxic effect of the cryoprotective agent, unlike the present invention. However, searching for a similar shelf-life effect, a preferred embodiment of the invention describes a last layer of hypoxic medium.
The problem of the art is then to provide a freezing method that could result in better post-thawing cell viability, in the save of time at tests performance and in diminish contamination risks. The solution provided by the present invention is a sequential freezing method resulting in a double or triple layered body container, which includes at least one fresh diluent layer. This method ease the thawing process in one single step, increases the thawing efficiency in a minimum of 15% and avoids the exchange of the viable cells to another container, thus diminishing contamination risks.